The present invention relates to canine and feline proteins. In particular, the present invention relates to feline interleukin-18 (IL-18), feline caspase-1 (casp-1), feline interleukin-12 (IL-12) single chain, and canine interleukin-12 (IL-12) single chain proteins and includes nucleic acid molecules encoding such proteins, antibodies raised against such proteins and/or inhibitors of such proteins or nucleic acid molecules. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and/or inhibitors, as well as their use to evaluate and regulate an immune response in an animal.
Regulation of immune and inflammatory responses in animals is important in disease management. Immune responses can be regulated by modifying the activity of immunoregulatory molecules and immune cells. Examples of such immunoregulatory molecules include IL-18, caspase-1 and IL-12. These molecules have been found to play a role in the treatment of several disorders including allergy, cancer, and pathogenic infection.
Monocytes and macrophages represent the first line of defense against disease. Various diseases and infections activate transcriptional and posttranslational events in monocytes and macrophages, which lead to the production of cytokines such as IL-18 and IL-12. These cytokines in turn activate responses in T and B cells helping to eliminate pathogens and/or disease in a animal. Both IL-18 and IL-12 augment cellular immunity by stimulating T cells to produce interferon gamma (IFN-xcex3) which inhibits the production of IgE formation without compromising B cell proliferation. IL-18, formerly referred to as interferon gamma inducing factor (IGIF), stimulates T cells to produce IFN-xcex3 and has been isolated from humans, dogs, and mice. A cDNA encoding human IL-18 was isolated and used to express recombinant human IL-18 by Ushio et al., 1996, J. Immunol.156, 4274-4279, GenBank accession number D49950. Feline IL-18 cDNA has a 85.8% homology to human IL-18 cDNA and feline IL-18 protein has a 81.7% homology to human IL-18 protein. A cDNA encoding canine IL-18 was isolated and used to express recombinant canine IL-18 by Okano et al., 1999, J. Interferon Cytokine Res. 19, 27-32, GenBank accession number Y11133. Feline IL-18 cDNA has a 90.7% homology to canine IL-18 cDNA and feline IL-18 protein has a 88.5% homology to canine IL-18 protein. A cDNA encoding murine IL-18 was isolated and used to express recombinant murine IL-18 by Okamura et al., 1995, Nature 378, 88-91, GenBank accession number D49949. Feline IL-18 cDNA has a 73.8% homology to murine IL-18 cDNA and feline IL-18 protein has a 70% homology to murine IL-18 protein. A cDNA encoding rat IL-18 was isolated by Culhane, et al. Mol. Psych. 3, 362-366 (1998), GenBank accession number AJ222813. Feline IL-18 cDNA has a 73.4% homology with rat IL-18 cDNA, and feline IL-18 protein has a 70.7% homology with rat IL-18 protein. A cDNA encoding equine IL-18 was isolated by Nicolson, et al. (unpublished, direct submission to GenBank, accession number Y11131). Feline IL18 cDNA has a 92% homology to equine IL-18 cDNA and feline IL-18 protein has a 89% homology to equine IL-18 protein. A cDNA encoding pig IL-18 was isolated by Penha-Goncalves, et al. (unpublished, direct submission to GenBank, accession number Y1 1132. Feline IL-18 cDNA has a 90.2% homology to pig IL-18 cDNA and feline IL-18 protein has a 85.9% homology to pig IL-18 protein. Expression of active IL-18 is controlled by caspase-1(IL-1 xcex2 converting enzyme). That is, IL-18 lacks a signal peptide so the precursor form of IL-18 (pro IL-18) is cleaved by caspase-1 resulting in a mature protein that is biologically active.
IL-12 is a heterodimer comprised of two subunits p40 and p35 which are covalently linked by a disulfide bond to form an active molecule. Simultaneous expression of the two subunits is necessary for the production of the biologically active heterodimer. Both human and murine p35 and p40 IL-12 single chain proteins (i.e., a single protein containing both p35 and p40 subunits) have been produced; see e.g., Lieschke et al., 1997, Nature Biotechnology 15, 35-40. Co-expression of the human and murine p35 and p40 cDNA subunits of IL-12 resulting in a biologically active IL-12 heterodimer was achieved by Gubler et al., 1991, Proc. Natl. Acad. Sci. U.S.A., 88, 4143-4147 and Schoenhaut et al., 1992, J. Immunol., 148, 3433-3440, respectively. cDNAs encoding canine IL-12 p35 and p40 subunits were isolated and co-transfected to express canine IL-12 by Okano et al., 1997, J. Interferon Cytokine Res. 17, 713-718. cDNAs encoding feline p35 and p40 subunits have been isolated and expressed; see, for example, Fehr et al., 1997, DNA Seq. 8, 77-82; Schijns et al., 1997, Immunogenetics 45, 462-463; Bush et al., 1994, Molec. Immunol. 31, 1373-1374. At the amino acid level, canine and feline IL-12 p40 subunit share 92.7 percent identity to each other; share 84.8 and 84.2 percent identity to human IL-12 p40, respectively; and share 67.4 and 68.9 percent identity to murine IL-12 p40, respectively. IL-12 shares some biological activities with IL-18 including IFN-xcex3 production in T cells. IL-18 and IL-12 in combination work synergistically to increase IFN-xcex3 production in T cells; as such these cytokines when utilized alone or in combination can be very effective in mediating IgE responses.
Caspase-1 may play a key role in the processing of IL-18 precursor in cells where IL-18 is produced. It may be that coexpression of caspase-1 along with IL-18 may be necessary for the proper processing of the IL-18 precursor and enhanced secretion of the processed IL-18 mature polypeptide. A cDNA encoding equine caspase-1 was isolated by Wardlow, et al. (unpublished; direct submission to GenBank, accession number AF090119). Feline caspase-1 cDNA has a 71% homology to equine caspase-1 cDNA and feline caspase-1 protein has a 48.8% homology to equine caspase-1 protein. A cDNA encoding equine caspase-1 was isolated by Cerretti, et al. (Science 256, p 97-100 (1992); GenBank accession number M87507). Feline caspase-1 cDNA has a 60% homology to human caspase-1 cDNA and feline caspase-1 protein has a 60% homology to human caspase-1 protein. A cDNA encoding rat caspase-1 (called interleukin-1 beta converting enzyme) was isolated by Keane, et al. (Cytokine 7(2) 105-110 1995); GenBank accession number U14647). Feline caspase-1 cDNA has a 55.4% homology to rat caspase-1 cDNA and feline caspase-1 protein has a 40.2% homology to rat caspase-1 protein. A cDNA encoding murine caspase-1 was isolated by Molineaux, et al. (Proc Natl. Acad. Sci 90, 1809-1813, 1993); GenBank accession number L28095). Feline caspase-1 cDNA has a 55.7% homology to murine caspase-1 cDNA and feline caspase-1 protein has a 38.5% homology to murine caspase-1 protein. A cDNA encoding canine caspase-1 was isolated by Taylor, et al. (2000) DNA Seq. 10(6), pp 387-394; GenBank accession number AF135967). Feline caspase-1 cDNA has a 90% homology to canine caspase-1 cDNA.
To date, however, neither IL-18 nor caspase-1, nor the nucleic acid molecules encoding such proteins, have been isolated from cats. Neither have IL-12 single chain proteins been produced using feline or canine IL-12 subunits. As such there remains a need for compounds and methods to regulate an immune response in cats and dogs through manipulation of IL-1 8, caspase-1 and IL-12 single chain activities.
The present invention relates to canine and feline proteins, nucleic acid molecules encoding such proteins, antibodies raised against such proteins and/or inhibitors of such proteins or nucleic acid molecules. In a preferred embodiment, the present invention relates to feline interleukin-18 (IL-1 8), feline caspase-1 (casp-1), feline interleukin-12 (IL-12) single chain and canine interleukin-12 single chain proteins, nucleic acid molecules, antibodies and inhibitors. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies and/or inhibitors, as well as their use to evaluate and regulate an immune response in an animal.
One embodiment of the present invention is an isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule selected from the group consisting of (i) a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ ID NO:11, and SEQ ID NO:13; and (ii) a nucleic acid molecule comprising at least 70 contiguous nucleotides identical in sequence to at least 70 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, and SEQ ID NO:41; (b) an isolated nucleic acid molecule selected from the group consisting of (i) a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:25, and (ii) a nucleic acid molecule comprising at least 70 contiguous nucleotides identical in sequence to at least 70 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:25; (c) an isolated nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule comprising ((a)) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:26, SEQ ID NO:29, and a nucleic acid sequence comprising at least 44 contiguous nucleotides identical in sequence to at least 44 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:26 and SEQ ID NO:29; ((b)) a nucleic acid linker of (XXX)n wherein n=0 to 60; and ((c)) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:32, SEQ ID NO:35, and a nucleic acid molecule comprising at least 44 contiguous nucleotides identical in sequence to at least 44 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:35, such that said nucleic acid molecule of (i) encodes a feline IL-12 single chain protein; and (ii) a nucleic acid molecule comprising a nucleic acid sequence fully complementary to the coding strand of any of said nucleic acid molecules as set forth in (i); (d) an isolated nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule comprising ((a)) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:52 and SEQ ID NO:58, and a nucleic acid sequence comprising at least 47 contiguous nucleotides identical in sequence to at least 47 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:46 and SEQ ID NO:49; ((b)) a nucleic acid linker of (XXX)n wherein n=0 to 60; and ((c)) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:49, and a nucleic acid molecule comprising at least 47 contiguous nucleotides identical in sequence to at least 47 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:46 and SEQ ID NO:49, such that said nucleic acid molecule of (i) encodes a canine IL-12 single chain protein; and (ii) a nucleic acid molecule comprising a nucleic acid sequence fully complementary to the coding strand of any of said nucleic acid molecules as set forth in (i); (e) an isolated nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule having a nucleic acid sequence that is at least 92 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:9, SEQ ID NO:41, SEQ ID NO:11, and SEQ ID NO:13; and (ii) a nucleic acid molecule comprising a fragment of a nucleic acid molecule of (i) wherein said fragment is at least 80 nucleotides in length; (f) an isolated nucleic acid molecule selected from the group consisting of (i) a nucleic acid molecule having a nucleic acid sequence that is at least 85 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:25, and (ii) a nucleic acid molecule comprising a fragment of a nucleic acid molecule of (i) wherein said fragment is at least 85 nucleotides in length; (g) an isolated nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule comprising ((a)) a nucleic acid molecule comprising a nucleic acid sequence that is at least 87 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:26 and SEQ ID NO:29, or a fragment thereof of at least 55 nucleotides in length; ((b)) a nucleic acid linker of (XXX)n wherein n=0 to 60; and ((c)) nucleic acid molecule comprising a nucleic acid sequence that is at least 87 percent identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:35, or a fragment thereof of at least 55 nucleotides in length, such that said nucleic acid molecule (i) encodes a feline IL-12 single chain protein; and (ii) a nucleic acid molecule comprising a nucleic acid sequence fully complementary to the coding strand of a nucleic acid molecule as set forth in (i); and (h) an isolated nucleic acid molecule selected from the group consisting of: (i) a nucleic acid molecule comprising ((a)) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:52 and SEQ ID NO:58, and a nucleic acid sequence comprising at least 55 contiguous nucleotides identical in sequence to at least 55 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of NO:52 and SEQ ID NO:58; ((b)) a nucleic acid linker of (XXX)n wherein n=0 to 60; and ((c)) an isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:49, and a nucleic acid molecule comprising at least 55 contiguous nucleotides identical in sequence to at least 55 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:35, such that said nucleic acid molecule of (i) encodes a canine IL-12 single chain protein; and (ii) a nucleic acid molecule comprising a nucleic acid sequence fully The present invention also includes recombinant molecules, recombinant viruses and recombinant cells comprising such IL-18, caspase-1, and IL-12 single chain nucleic acid molecules and methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention is an isolated nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule having a nucleic acid sequence encoding an IL-18 protein selected from the group consisting of: (i) a protein selected from the group consisting of ((a)) a protein having an amino acid sequence that is at least 92 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ I) NO:5, SEQ ID NO:8 and SEQ ID NO:12, and ((b)) a protein comprising a fragment of a protein of ((a)), wherein said fragment is at least 30 amino acids in length; and (ii) a protein comprising at least 25 contiguous amino acids identical in sequence to at least 25 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:12; (b) a nucleic acid molecule having a nucleic acid sequence encoding a caspase-1 protein selected from the group consisting of: (i) a protein selected from the group consisting of ((a)) a protein having an amino acid sequence that is at least 85 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24, and ((b)) a protein comprising a fragment of a protein of ((a)), wherein said fragment is at least 30 amino acids in length; and (ii) a protein comprising at least 25 contiguous amino acids identical in sequence to at least 25 contiguous amino acids selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24; (c) a nucleic acid molecule having a nucleic acid sequence encoding an IL12 single chain protein comprising an IL-12 p40 subunit domain linked to a IL12 p35 subunit domain, wherein said p40 subunit domain is selected from the group consisting of (i) a p40 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30, (ii) a p40 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 30 amino acids in length, and (iii) a p40 subunit protein comprising at least 23 contiguous amino acids identical in sequence to at least 23 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30 and wherein said p35 subunit domain is selected from the group consisting of (i) a p35 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36, (ii) a p35 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 30 amino acids in length, and (iii) a p35 subunit protein comprising at least 23 contiguous amino acids identical in sequence to at least 23 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36; (d) a nucleic acid molecule having a nucleic acid sequence encoding an IL-12 single chain protein comprising an IL-12 p40 subunit domain linked to a IL-12 p35 subunit domain, wherein said p40 subunit domain is selected from the group consisting of (i) a p40 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ ID NO:59, (ii) a p40 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 40 amino acids in length, and (iii) a p40 subunit protein comprising at least 31 contiguous amino acids identical in sequence to at least 31 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ ID NO:59, and wherein said p35 subunit domain is selected from the group consisting of (i) a p35 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:50, (ii) a p35 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 40 amino acids in length, and (iii) a p35 subunit protein comprising at least 31 contiguous amino acids identical in sequence to at least 31 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:50; (e) a nucleic acid molecule comprising a nucleic acid sequence fully complementary to the coding strand of any of said nucleic acid molecules as set forth in (a), (b), (c), or (d). The present invention also includes recombinant molecules, recombinant viruses and recombinant cells comprising such IL-18, caspase-1, and IL-12 single chain nucleic acid molecules and methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
Another embodiment of the present invention is an isolated protein selected from the group consisting of: (a) an isolated IL-18 protein selected from the group consisting of: (i) an isolated protein of at least 25 amino acids in length, wherein said protein has an at least 25 contiguous amino acid region identical in sequence to a 25 contiguous amino acid region selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:12; and (ii) an isolated protein having an amino acid sequence that is at least 92 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:12 and a fragment thereof of at least 30 nucleotides; wherein said isolated protein has a function selected from the group consisting of (i) eliciting an immune response against an IL-18 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 and SEQ ID NO:12, (ii) selectively binding to an antibody raised against an IL-18 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, and SEQ ID NO:12, and (iii) exhibiting IL-18 activity; (b) an isolated caspase-1 protein selected from the group consisting of: (i) an isolated protein of at least about 25 amino acids in length, wherein said protein has an at least 25 contiguous amino acid region identical in sequence to a 25 contiguous amino acid region selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24; and (ii) an isolated protein having an amino acid sequence that is at least 85 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24 and has a nucleic acid fragment thereof of at least 30 nucleotides; wherein said isolated protein has a function selected from the group consisting of (i) eliciting an immune response against a caspase-1 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24, (ii) ii) selectively binding to an antibody raised against caspase-1 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, and SEQ ID NO:24, and (iii) exhibiting caspase-1 activity; (c) an isolated IL-12 single chain protein comprising an IL-12 p40 subunit domain linked to an IL-12 p35 subunit domain, wherein said p40 subunit domain is selected from the group consisting of (i) a p40 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30, (ii) a p40 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 30 amino acids in length, and (iii) a p40 subunit protein comprising at least 23 contiguous amino acids identical in sequence to at least 23 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30 and wherein said p35 subunit domain is selected from the group consisting of (i) a p35 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36, (ii) a p35 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 30 amino acids in length, and (iii) a p35 subunit protein comprising at least 23 contiguous amino acids identical in sequence to at least 23 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36; wherein said isolated protein has a function selected from the group consisting of (i) eliciting an immune response against an IL-12 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:39 and SEQ ID NO:44, (ii) selectively binding to an antibody raised against an IL-12 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:59, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:62, and/or SEQ ID NO:67, and (iii) exhibiting IL-12 activity; and (d) an isolated IL-12 single chain protein comprising an IL-12 p40 subunit domain linked to an IL-12p35 subunit domain, wherein said p40 subunit domain is selected from the group consisting of (i) a p40 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ ID NO:59, (ii) a p40 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 40 amino acids in length, and (iii) a p40 subunit protein comprising at least 31 contiguous amino acids identical in sequence to at least 31 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:53 and SEQ I) NO:59; wherein said p35 subunit domain is selected from the group consisting of (i) a p35 subunit protein having an amino acid sequence that is at least 84 percent identical to an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:50, (ii) a p35 subunit protein comprising a fragment of a protein of (i), wherein said fragment is at least 40 amino acids in length, and (iii) a p35 subunit protein comprising at least 31 contiguous amino acids identical in sequence to at least 31 contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:50; and wherein said isolated protein has a function selected from the group consisting of (i) eliciting an immune response against an IL-12 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:44, SEQ ID NO:62, and SEQ ID NO:67, (ii) selectively binding to an antibody raised against an IL-12 protein having an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, and SEQ ID NO:59, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:62, and/or SEQ ID NO:67, and (iii) exhibiting IL-12 activity. The present invention also includes recombinant molecules, recombinant viruses and recombinant cells comprising such IL-18, caspase-1, and IL-12 single chain nucleic acid molecules and methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
The present invention also includes an antibody that selectively binds to a protein of the present invention as well as methods to produce and use such proteins or antibodies. By selectively is meant an antibody that binds to a protein of the present invention, but does not bind a similar protein of another species.
One aspect of the present invention is a therapeutic composition that, when administered to an animal, regulates an immune response in the animal. Such a therapeutic composition includes at least one of the following protective compounds: an IL-18, caspase-1, or IL-12 single chain protein of the present invention, a mimetope of any of the proteins, a multimeric form of any of the proteins, an isolated nucleic acid molecule of the present invention, an antibody that selectively binds any of the proteins, and/or an inhibitor of a protein activity identified by its ability to inhibit the activity of any of the proteins. Also included is a method to regulate an immune response by administering such a therapeutic composition to an animal.
The present invention also includes a method to produce a protein of the present invention; such a method includes the step of culturing a recombinant cell capable of expressing a protein being encoded by a nucleic acid molecule of the present invention.
Another embodiment of the present invention is a method to identify a compound capable of regulating an immune response in an animal, a method selected from the group consisting of: (a) contacting an isolated feline IL-18 protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has T cell stimulating activity inducing T cells to make interferon gamma, and determining if the putative inhibitory compound inhibits that activity; (b) contacting an isolated feline caspase-1 protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein cleaves precursor form of IL-18 resulting in biologically active IL-18, and determining if the putative inhibitory compound inhibits that activity; and (c) contacting an isolated IL-12 single chain protein with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has T cell proliferation stimulating activity, and determining if the putative inhibitory compound inhibits that activity.